Improving the delivery of drugs and other agents to target cells and tissues has been the focus of considerable research for many years. Though many attempts have been made to develop effective methods for importing biologically active molecules into cells, both in vivo and in vitro, none has proved to be entirely satisfactory. Optimizing the association of the drug with its intracellular target, while minimizing intercellular redistribution of the drug, e.g., to neighboring cells, is often difficult or inefficient. Accordingly, a major goal has been to develop methods and compositions for specifically targeting agents to cells and tissues. Benefits of such treatment include avoiding the general physiological effects of inappropriate delivery of such agents to other cells and tissues, such as uninfected cells.
Intracellular targeting may be achieved by methods and compositions that allow accumulation or retention of biologically active agents inside cells.
Assay methods capable of determining the presence, absence or amounts of an infectious agent or presence or absence of a medical condition are of practical utility in the search for inhibitors of such an agent or condition.
There is a need for therapeutic agents, e.g., prodrugs, having desired pharmacokinetic properties, including enhanced activity, improved oral bioavailability, greater potency and extended effective half-life in vivo. Identified prodrugs will preferably have fewer side effects, less complicated dosing schedules, and be orally active. Such prodrugs may be useful to limit the establishment and progression of a medical condition as well as in diagnostic assays for a medical condition. As such, a need exists for enzymes that facilitate the identification of such prodrugs.
There is consensus that the bioactivation of phosphoramidate prodrugs such as nucleotide amidate triesters may follow a general scheme (Valette et al., J. Med. Chem., 39: 1981-1991 (1996); McGuigan et al., Antivir. Chem. Chemotheraphy, 9: 109-115 (1998), McGuigan et al., Antivir. Chem. Chemotheraphy, 9: 473-479 (1998); Saboulard et al., Mol. Pharmacol., 56: 693-704 (1999); Siddiqui et al., J. Med. Chem., 42: 4122-4128 (1999)). See FIG. 1. Step A is the hydrolysis of the amino acid-like carboxylic ester. A nucleophilic attack by the carboxylic acid of the phosphorous (Step B) is believed to initiate the formation of a 5-membered cyclic intermediate, which intermediate is quickly hydrolyzed to the monoamidate diester (referred to as the amino acid nucleoside monophosphate, AAM, Metabolite X). AAM compounds such as Metabolite X are considered intracellular depot forms, for example of antiviral nucleoside. Various enzymes as well as non-enzymatic catalysis have been implicated in the hydrolysis of the amide bond of AAM compounds resulting in the formation of the nucleotide. The nucleotide is activated by enzymatic phosphorylation to nucleotide di- and tri-phosphates. Ester hydrolase activity might also be hypothesized to apply to prodrug molecules other than phosphoramidates. However, until now identification of the mechanisms and specificities of ester hydrolase cleavage of prodrugs has been constrained by the limited availability of identifiable ester hydrolase enzymes.